Although the factors that regulate SOD1 entrance, processing and aggregation within mitochondria are not fully understood, copper chaperone for SOD1 (CCS), appears to be an important determinant of SOD1 presence within mitochondria. In vitro evidence suggests that direct CCS interaction with SOD1 facilitates the conversion of immature apo-SOD1, that is capable of transit into mitochondria, to a mature holo-SOD1 form that is retained within mitochondria. Whether over-expressing CCS in vivo will alter the sub-cellular distribution of SOD1 in a way that favors intra-mitochondrial SOD1 retention and consequently impact mutant SOD1 induced disease is unknown. To address these key questions of SOD1 biochemistry in vivo, we have generated transgenic mice expressing human CCS in high levels within the CNS and crossed them to G93A-SOD1 or wild type (WT) SOD1 transgenic mice. Both CCS transgenic mice and CCS/WT-SOD1 dual transgenic mice are neurologically normal. In contrast, CCS/G93A-SOD1 dual transgenic mice develop accelerated neurological deficits, with a mean survival of 36 days compared to 242 days for G93A-SOD1 mice. Immuno-electron microscopy and sub-cellular fractionation studies on spinal cord show that G93A-SOD1 is enriched within mitochondria in the presence of CCS over-expression. Our results indicate that CCS over-expression in G93A-SOD1 mice produces severe mitochondrial pathology and accelerates disease course. The extent of these changes tells us that over-expressing CCS is changing a fundamental principle of G93A SOD1 induced neurological disease and raises two central questions. How is G93A SOD1 altered by CCS over-expression? How does this change in G93A SOD1 lead to the severe mitochondrial phenotype? Answering these two questions forms the basis for this grant proposal. Completion of the proposed aims in this grant will provide insights into the cellular and molecular mechanisms underlying one form of familial amyotrophic lateral sclerosis (ALS) related to mutations in the copper, zinc superoxide dismutase gene.